Mechanisms involved in retrotranslocation of ricin from the endoplasmic reticulum to the cytosol

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Abstract

Newly synthesised proteins are therefore subjected to a strict control machinery in the ER lumen where different chaperones facilitate folding during and after translocation. If the folding process is incomplete, misfolded proteins are retrotranslocated (or dislocated) from the ER to the cytosol where they are finally degraded by the proteasomes. The process of selective protein export from the ER to the cytosol for proteasomal degradation is known as ER-associated degradation (ERAD).

The Der1-protein of the budding yeast, Saccharomyces cerevisiae (S. Cerevisiae) has been shown to be required for retrotranslocation and degradation of a subset of ER substrates. The mammalian genome contains three Der1-like proteins, named Derlin-1, -2 and -3, and the amount of substrates dependent on Derlin proteins for retrotranslocation to the cytosol is emerging.

The protein toxin ricin is a useful tool to study intracellular transport mechanisms. It consists of an A- and a B-chain that are linked together by a disulfide-bond. The B-chain is a lectin that binds to galactose-containing surface-receptors and the A-chain is able to bind to and inactivate ribosomes. The whole toxin is endocytosed and is transported in a retrograde manner via the trans-Golgi network (TGN) to the ER before it is retrotranslocated to the cytosol. The disulfide-bond can be reduced by protein disulfide isomerase (PDI) and thioredoxin reductase in the ER. It was proposed that this event partially unfolds the A-chain, making it able to cross the ER-membrane, presumably through the Sec61 translocon. However, it has previously been shown that a ricin molecule with a non-reducible bond is equally cytotoxic as native ricin, indicating that intact ricin (holotoxin) might somehow reach the cytosol. Ricin holotoxin binds to both ER degradation enhancing α-mannosidase I-like protein (EDEM) and Sec61 and preliminary data from our laboratory suggests that holotoxin is also able to reach the cytosol.

In the present study, we aimed to obtain new insight into the mechanisms involved in targeting of ricin to the cytosol. We have focused on the role of Derlin-3 in retrotranslocation of ricin, but have also addressed some questions regarding transport of holotoxin to the cytosol.